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Monoclonal antibody and ELISA method and kit for detecting carbadox and cydox metabolites

A monoclonal antibody and metabolite technology, applied in the field of veterinary drug residue analysis and immunology, to achieve high accuracy, save time and cost, and minimize health hazards

Inactive Publication Date: 2017-07-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, there are few reports on the detection methods of quinoxaline deoxygenated metabolites, so a monoclonal antibody with good specificity and high sensitivity was established to detect the metabolism of carbadox and cydox in animals or meat. product appears to be necessary

Method used

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  • Monoclonal antibody and ELISA method and kit for detecting carbadox and cydox metabolites
  • Monoclonal antibody and ELISA method and kit for detecting carbadox and cydox metabolites
  • Monoclonal antibody and ELISA method and kit for detecting carbadox and cydox metabolites

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Experimental program
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Embodiment 1

[0030] The preparation of embodiment 1 immunogen and coating former

[0031] 1.1 Preparation of immunogen QHCPA and bovine serum albumin conjugate (QHCPA-HSA)

[0032] 1.1.1 The synthesis of 2-methylquinoxaline refers to the synthetic method of Xu et al. (2006), and makes improvements. Take 1.08g (0.01mol) of o-phenylenediamine and put it into a 500mL three-necked flask, add 150mL of methanol, heat up to 70°C, reflux, stir to dissolve it; take 0.90mL (0.01mol) of aceglyoxal, add 10mL of methanol Dilute it, add it dropwise to the above solution, and detect the reaction process by thin-layer chromatography, using petroleum ether: ethyl acetate = 3:1 as the developer. Reacted for 12 hours, filtered hot, distilled off a small amount of methanol, and recrystallized to obtain 1.27 g of light yellow needle-point crystals, namely 2-methylquinoxaline, with a yield of 70.5%. The reaction equation is as follows:

[0033]

[0034] 1.1.2 Synthesis of 2-formylquinoxaline Take 1.1g of ...

Embodiment 2

[0047] The preparation of embodiment 2 monoclonal antibody

[0048] Preparation of hybridoma cells: referring to Yang Hanchun's "Animal Immunology", the immunogen QHCPA-human serum albumin conjugate (QHCPA-HSA) prepared in Example 1 was used to immunize Balb / C mice (purchased from Experimental Animal Center). The immunization procedure is as follows: for basic immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant is injected subcutaneously at the back of the neck of the mouse; Protein emulsion of immunogen for booster immunization. From the third immunization, the tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was detected by indirect ELISA method. Immunization qualified mice (high titer, good sensitivity) stop immunization in preparation for fusion. At the time of fusion, take a Balb / C mouse that has undergone the final booster imm...

Embodiment 3

[0053] Example 3 Establishment of ELISA detection method for indirect competition between desdioxycarbazine and desdioxyridine sedox

[0054] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0055] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0056] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0057] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, Tween 200.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0058] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0059] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0060] Substrate B: Na 2 HPO 4 14.6g, citric...

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Abstract

The invention discloses a specificity monoclonal antibody capable of distinguishing various carbadox and cyadox metabolism products and an enzyme linked immunosorbent assay method and kit for detecting the carbadox and cyadox metabolism products. According to the invention, the monoclonal antibody is secreted by a hybridoma cell strain DCBX5B1 of which the preservation number is CCTCC NO: C201495. Compared with the prior art, the monoclonal antibody disclosed by the invention can distinguish DCBX, bisdesoxy cyadox and desoxy cyadox (N4) at the same time, and has higher sensitivity and specificity. The ELISA method and the kit, disclosed by the invention, can detect the residue of the carbadox and cyadox metabolism products in meat food at the same time, and have the advantages of high detection efficiency, high sensitivity, high precision, high accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and relates to a monoclonal antibody capable of recognizing carbadox and cycadol metabolites and an enzyme-linked immunosorbent method (ELISA) for detecting carbadox and cycadol metabolites. ) and kits. Background technique [0002] Dedioxycarboxylate and dedioxycydol are quinoxaline drugs, which are the products of the deoxygenation and metabolism of the prototype drugs carbadox and cyadox in animals. structure, carbadox and quincedo have a broad antibacterial spectrum, and their anti-gram-negative bacteria are stronger than positive bacteria. It has been widely used in veterinary clinic and breeding industry. People abuse drugs in pursuit of economic benefits, resulting in a large amount of drug residues in animal food. Quinoxaline drugs can cause toxic and side effects such as tertiary effects, photosensitivity, and adrenal cortex damage to animals. S...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/531C12R1/91
Inventor 袁宗辉何秀平彭大鹏潘源虎王玉莲陈冬梅陶燕飞刘振利
Owner HUAZHONG AGRI UNIV
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